Studies will be continued on the overall topography, sequence location and nature of the agonist recognition and the non-competitive inhibitor sites on the nicotinic acetylcholine receptor. Two general approaches will be employed: site specific labeling and fluorescence energy transfer. With certain compounds this approach becomes complementary and we have synthesized or characterized a series of agents for this purpose. In general, fluorescence studies yield information on intersite distances, molecular motion of the bound ligand and conformational changes associated with binding. Site specific labeling reveals information on the amino acid residues contributing to the binding site. The fluorescence energy transfer and labeling studies with fluorophores will entail cross-linking alpha-toxin to the receptor at the location of the fluorophore and linking covalent analogues of certain fluorophores with the receptor. This should define amino acid residues contributing to the respective binding surfaces. A series of compounds, lophotoxin, p-(trimethylammonium) benzene diazonium fluoroborate, and azidoethidium will be used to label the agonist and non- competitive inhibitor sites, respectively. Lophotoxin is the most novel of the compounds for its diterpenoid structure is devoid of nitrogens and a positive charge. Lophotoxin reacts selectively with tyrosine 190 in the alpha-subunit and we propose to characterize this reaction mechanism and bond of conjugation. Several naturally occurring receptors with unique sequence characteristics as well as heterologous expression systems are projected for study. The ligands under study should prove useful in neuronal and muscle nicotinic receptor subtyping. Some are currently being investigated for the treatment of muscle spasms (blepharospasm of the eye).